Medium for culturing normal human epidermal melanocytes

ABSTRACT

Provided is a medium for culturing normal human epidermal melanocytes in vitro. The medium comprises a basal medium for culturing animal cells, Ca 2+   at a final concentration of between about 0.15 mM and about 1.2 mM and Mg 2+   at a final concentration of 1.2 mM and 6 mM or Ca 2+   at a final concentration of between about 0.9 mM and about 1.2 mM and Mg 2+   at a final concentration between about 0.6 mM and 6.0 mM and 0.001 to 0.1% scrum (v/v). The medium can promote both dendrite formation and proliferation of the cells.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a medium suitable for in vitro culturing normal human epidermal melanocyte.

2. Prior Art

In vitro culturing of normal cells derived from human is a key technique for investigation in many fields including medical and pharmaceutical, or development of drug, cosmetics and the like. Culturing of normal human melanocyte in vitro is a useful steps for studying, such as biosynthesis of melanine in human skin or whitening of the skin. In order to culture said cells, known basal media for culturing animal cells such as MCDB153 or M199 containing supplements, for example growth factors, such as epidermal growth factor and fibroblast growth factor, hormones such as insulin and triiodothyronine, carriers such as cholera toxin and transferrin, bovine pituitary extract, bovine hypothalamus extract and fetal bovine serum (FBS).

However, those conventional media cannot provide enough growth without comparatively large amount of serum. In addition, while the morphology of melanocytes in vivo characterized by dendrite formation and ramification, high population of melanocyte cultured in vitro with a conventional media shows quite different such as bipolar morphology. Dendrite formation of in vitro cultured cells can be promoted by adding an agent that increase intracellular levels of cAMP, such as dbcAMP or IBMX. However, these agents cause suppression of cell proliferation (K, Nakazawa et. al., Pigment Cell Research (1993) 6, 406-416).

SUMMARY OF THE INVENTION

An object of the present invention is to provided a medium for in vitro culturing normal human epidermal melanocyte which can promote cell proliferation.

In another object of the present invention is to provide a medium for in vitro culturing normal human epidermal melanocyte which can promote both proliferation and dendrite formation of said cells.

In further object of the present invention is to provide a medium for in vitro culturing normal human epidermal melanocyte which can promote intracellular melanin synthesis of said cells.

According to one aspect of the present invention, there is provided a medium comprising of a basal medium for culturing animal cells, Ca²⁺ at a final concentration of between about 0.9 mM and about 1.2 mM and Mg²⁺ at a final concentration of between about 0.6 mM and 6 mM. In addition, the present invention also provides a medium useful for culturing normal human epidermal melanocyte, comprising of a basal medium for culturing animal cells, Ca²⁺ at a final concentration of between about 0.15 mM and about 1.2 mM and Mg²⁺ at a final concentration of between about 1.2 mM and 6 mM. The most preferable medium according to the present invention contains Ca²⁺ at a final concentration of between about 0.9 mM and about 1.2 mM and Mg²⁺ at a final concentration of between about 1.2 mM and 6 mM.

The medium may further comprise one or more growth factor useful for growth of human melanocyte. The medium of the present invention may still further comprise biological materials such as bovine pituitary extract.

The medium of the present invention may further comprise between about 0.001% and about 0.1% (v/v) of heat inactivated fetal bovine serum.

In another aspect of the present invention, there is provided a method for culturing normal human epidermal melanocyte which contains culturing said cells in the medium of the present invention under cell-culturing conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents results of example 1.

FIG. 2 represents results of example 2.

FIG. 3 represents results of example 3.

FIG. 4 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.15 mM of Ca²⁺ and 0.6 mM of Mg²⁺ at 7 days after inoculation. Original magnification×40.

FIG. 5. is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.15 mM of Ca²⁺ and 1.8 mM of Mg²⁺ at 7 days after inoculation in a medium. Original magnification×40.

FIG. 6 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.9 mM of Ca²⁺ and 0.6 mM of Mg²⁺ at 7 days after inoculation. Original magnification×40.

FIG. 7 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.9 mM of Ca²⁺ and 1.8 mM of Mg²⁺ at 7 days after inoculation. Original magnification×40.

FIG. 8 is a photomicrograph of normal human epidermal melanocyte cultured in a medium containing 0.9 mM of Ca²⁺ and 6.0 mM of Mg²⁺ at 7 days after inoculation. Original magnification×40.

FIG. 9 represents results of example 4.

FIG. 10 represents results of example 5.

FIG. 11 represents results of example 6.

FIG. 12 represents results of example 7.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The inventors found that by controlling Ca²⁺ and Mg²⁺ concentration within a defined range, a basal medium for animal cell culture can be used for in vitro culture of normal human epidermal melanocyte. The medium of the present invention promotes both dendrite formation and proliferation of melanocytes. In addition, melanocyte cultured with the medium of the present invention shows comparatively dark color indicating that the cells can synthesis and deposit comparatively large amount of melanin.

"Basal medium for culturing animal cells" used in the present invention may be any known nutrient medium which is suitable for in vitro culture of normal human epidermal melanocyte. Examples of preferred media include MCDB 153, M199, Eagle's MEM, Dulbecco's modified of Eagle's MEM, HAM F 12, RPMI1640 and the like. Most preferable basal medium is MCDB 153 or modified MCDB 153 which contains enriched amino acid ingredients than standard MCDB 153. In the following table 1 and 2, there are shown the ingredients of standard MCDB 153 and modified MCDB 153. In the present specification, without specific indication, "MCDB 153" represents not only standard MCDB 153 of table 1 but also modified MCDB 153 of table 2. The modified MCDB 153 is identical to the standard MCDB 153 except for containing enriched L-histidine, L-isoleucine, L-methionine HCl, L-phenylalanine, L-tryptophane, and L-tyrosine and supplemented with ethanol amine and phosphoethanol amine.

                  TABLE 1     ______________________________________     Components of standard MCDB 153:     component       mol/l     mg/l     ______________________________________     L-Alanine       1.0 × 10.sup.-4                               8.91     L-Arginine HCl  1.0 × 10.sup.-3                               210.7     L-Asparagine.H.sub.2 O                     1.0 × 10.sup.-4                               15.01     L-Aspartic Acid 3.0 × 10.sup.-5                               3.99     L-Cysteine HCl.H.sub.2 O                     2.4 × 10.sup.-4                               42.04     L-Glutamic Acid 1.0 × 10.sup.-4                               14.71     L-Glutamine     6.0 × 10.sup.-3                               877.2     Glycine         1.0 × 10.sup.-4                               7.51     L-Histidine HCl.H.sub.2 O                     8.0 × 10.sup.-5                               16.77     L-Isoleucine    1.5 × 10.sup.-5                               1.968     L-Leucine       5.0 × 10.sup.-4                               65.6     L-Lysine HCl    1.0 × 10.sup.-4                               18.27     L-Methionine HCl                     3.0 × 10.sup.-5                               4.476     L-Phenylalanine 3.0 × 10.sup.-5                               4.956     L-Proline       3.0 × 10.sup.-4                               34.53     L-Serine        6.0 × 10.sup.-4                               63.06     L-Threonine     1.0 × 10.sup.-4                               11.91     L-Tryptophan    1.5 × 10.sup.-5                               3.06     L-Tyrosine      1.5 × 10.sup.-5                               2.718     L-Valine        3.0 × 10.sup.-4                               35.13     d-Biotin        6.0 × 10.sup.-8                               0.0146     Folic Acid      1.8 × 10.sup.-6                               0.79     DL-alpha-Lipoic Acid                     1.0 × 10.sup.-6                               0.2063     Niacinamide     3.0 × 10.sup.-7                               0.03663     D-Pantothenate(Ca)                     1.0 × 10.sup.-6                               0.468     Pyridoxine HCl  3.0 × 10.sup.-7                               0.6171     Riboflavin      1.0 × 10.sup.-7                               0.03764     Thiamin HCl     1.0 × 10.sup.-6                               0.3373     Vitamin B.sub.12                     3.0 × 10.sup.-6                               4.07     Adenine         1.8 × 10.sup.-4                               24.32     Choline Chloride                     1.0 × 10.sup.-4                               13.96     D-Glucose       6.0 × 10.sup.-3                               1081     myo-lnositol    1.0 × 10.sup.-4                               18.02     Putrescine 2HCl 1.0 × 10.sup.-6                               0.1611     Sodium Pyruvate 5.0 × 10.sup.-4                               55.0     Thymidine       3.0 × 10.sup.-6                               0.7266     CaCl.sub.2.2H.sub.2 O                     1.5 × 10.sup.-4                               22.05     KCl             1.5 × 10.sup.-3                               111.83     MgCl.sub.2.6H.sub.2 O                     6.0 × 10.sup.-4                               122.0     NaCl            1.3 × 10.sup.-1                               7599     Na.sub.2 HPO.sub.4.7H.sub.2 O                     2.0 × 10.sup.-3                               536.2     CuSO.sub.4.5H.sub.2 O                     1.1 × 10.sup.-8                               0.00275     FeSO.sub.4.7H.sub.2 O                     5.0 × 10.sup.-6                               1.39     H.sub.2 SeO.sub.3                     3.0 × 10.sup.-8                               0.003867     MnSO.sub.4.5H.sub.2 O                     1.0 × 10.sup.-9                               0.000241     Na.sub.2 SiO.sub.3.9H.sub.2 O                     5.0 × 10.sup.-7                               0.1421     (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                     1.0 × 10.sup.-9                               0.001236     NH.sub.4 VO.sub.3                     5.0 × 10.sup.-9                               0.000585     NiCl.sub.2.6H.sub.2 O                     .sup. 5.0 × 10.sup.-10                               0.000119     SnCl.sub.2.2H.sub.2 O                     .sup. 5.0 × 10.sup.-10                               0.000113     ZnSO.sub.4.7H.sub.2 O                     5.0 × 10.sup.-7                               0.1438     HEPES           2.8 × 10.sup.-2                               6600     NaHCO.sub.3     1.4 × 10.sup.-2                               1176     Sodium Acetate.3H.sub.2 O                     3.7 × 10.sup.-3                               500     Phenol Red(Na)  3.3 × 10.sup.-6                               1.242     ______________________________________

                  TABLE 2     ______________________________________     Components of modified MCDB 153:     Component       mol/l     mg/l     ______________________________________     L-Alanine       1.0 × 10.sup.-4                               8.91     L-Arginine HCl  1.0 × 10.sup.-3                               210.7     L-Asparagine.H.sub.2 O                     1.0 × 10.sup.-4                               15.01     L-Aspartic Acid 3.0 × 10.sup.-5                               3.99     L-Cysteine HCl.H.sub.2 O                     2.4 × 10.sup.-4                               42.04     L-Glutamic Acid 1.0 × 10.sup.-4                               14.71     L-Glutamine     6.0 × 10.sup.-3                               877.2     Glycine         1.0 × 10.sup.-4                               7.51     L-Histidine HCl.H.sub.2 O                     3.2 × 10.sup.-4                               67.08     L-Isoleucine    7.7 × 10.sup.-4                               100.3     L-Leucine       5.0 × 10.sup.-4                               65.6     L-Lysine HCl    1.0 × 10.sup.-4                               18.27     L-Methionine HCl                     1.2 × 10.sup.-4                               17.91     L-Phenylalanie  1.2 × 10.sup.-4                               19.82     L-Proline       3.0 × 10.sup.-4                               34.53     L-Serine        6.0 × 10.sup.-4                               63.06     L-Threonine     1.0 × 10.sup.-4                               11.91     L-Tryptophan    6.0 × 10.sup.-5                               12.25     L-Tyrosine      9.0 × 10.sup.-5                               16.31     L-Valine        3.0 × 10.sup.-4                               35.13     d-Biotin        6.0 × 10.sup.-8                               0.0146     Folic Acid      1.8 × 10.sup.-6                               0.79     DL-alpha-Lipoic Acid                     1.0 × 10.sup.-6                               0.2063     Niacinamide     3.0 × 10.sup.-7                               0.03663     Ethanolamine    1.0 × 10.sup.-4                               6.108     Phosphoethanolamine                     1.0 × 10.sup.-4                               14.106     D-Pantothenate(Ca)                     1.0 × 10.sup.-6                               0.468     Pyridoxine HCl  3.0 × 10.sup.-7                               0.06171     Riboflavin      1.0 × 10.sup.-7                               0.03764     Thiamin HCl     1.0 × 10.sup.-6                               0.3373     Vitamin B.sub.12                     3.0 × 10.sup.-6                               4.07     Adenine         1.8 × 10.sup.-4                               24.32     Choline Chloride                     1.0 × 10.sup.-4                               13.96     D-Glucose       6.0 × 10.sup.-3                               1081     myo-lnositol    1.0 × 10.sup.-4                               18.02     Putrescine 2HCl 1.0 × 10.sup.-6                               0.1611     Sodium Pyruvate 5.0 × 10.sup.-4                               55.0     Thymidine       3.0 × 10.sup.-6                               0.7266     CaCl.sub.2.2H.sub.2 O                     1.5 × 10.sup.-4                               22.05     KCl             1.5 × 10.sup.-3                               111.83     MgCl.sub.2.6H.sub.2 O                     6.0 × 10.sup.-4                               122.0     NaCl            1.3 × 10.sup.-1                               7599     Na.sub.2 HPO.sub.4.7H.sub.2 O                     2.0 × 10.sup.-3                               536.2     CuSO.sub.4.5H.sub.2 O                     1.1 × 10.sup.-8                               0.00275     FeSO.sub.4.7H.sub.2 O                     5.0 × 10.sup.-6                               1.39     H.sub.2 SeO.sub.3                     3.0 × 10.sup.-8                               0.003867     MnSO.sub.4.5H.sub.2 O                     1.0 × 10.sup.-9                               0.000241     Na.sub.2 SiO.sub.3.9H.sub.2 O                     5.0 × 10.sup.-7                               0.1421     (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                     1.0 × 10.sup.-9                               0.001236     NH.sub.4 VO.sub.3                     5.0 × 10.sup.-9                               0.000585     NiCl.sub.2.6H.sub.2 O                     .sup. 5.0 × 10.sup.-10                               0.000119     SnCl.sub.2.2H.sub.2 O                     .sup. 5.0 × 10.sup.-10                               0.000113     ZnSO.sub.4.7H.sub.2 O                     5.0 × 10.sup.-7                               0.1438     HEPES           2.8 × 10.sup.-2                               6600     NaHCO.sub.3     1.4 × 10.sup.-2                               1176     Sodium Acetate 3H.sub.2 O                     3.7 × 10.sup.-3                               500     Phenol Red(Na)  3.3 × 10.sup.-6                               1.242     ______________________________________

According to the present invention, at least one of Ca²⁺ and Mg²⁺ concentrations is enriched from the basal medium. The final Ca²⁺ concentration of the medium of the present invention is in the range between about 0.15 mM and about 1.2 mM, preferably, about 0.9 mM and about 1.2 mM. However, when Ca²⁺ concentration is lower than 0.9 mM, final concentration of Mg²⁺ is adjusted in the range of between about 1.2 mM and 1.8 mM. The final concentration of Ca²⁺ can be adjusted by adding Ca²⁺ source such as CaCl₂ into the basal medium.

The final Mg²⁺ concentration of the medium of the present invention is in the range between about 0.6 mM and about 6 mM, preferably, about 1.2 mM and about 1.8 mM. However, when Mg²⁺ concentration is lower than 1.2 mM, a final concentration of Ca²⁺ is adjusted to more than 0.9 mM. The final concentration of Mg²⁺ can be adjusted by adding Mg²⁺ source such as MgCl₂ and MgSO₄ into the basal medium.

The medium of the present invention may further comprise various supplements that are employed in conventional animal cell in vitro culture, if desired. For example, one or more growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), Interleukin-1, transforming growth factor alpha and the like, are preferably contained in the medium.

The medium of the present invention preferably contains comparatively small amount of serum. Examples for serum include that of derived from bovine, horse, porcine and the like. Among them, fetal bovine serum is most preferable because of its commercial availability. In the medium of the present invention, serum may be contained at a final concentration of between about 0.001% and about 1%(v/v), more preferably between about 0.001% and about 0.1% (v/v).

Further, the medium of the present invention may comprise biological materials for example bovine pituitary extract (BPE), bovine hypothalamus extract, bovine cerebrum extract and albumin; insulin, phorbol 12 myristrate 13-acetate(PMA), heparin, hydrocortisone, transferrin, cholera toxin, and triiodothyronine. Further more, the medium may comprise antibiotics such as gentamicin sulfate, amphotericin B, penicillin, mitomycin, kanamycin sulfate and streptomycin. These supplements described above may be added into the medium singly or in mixture as required.

The present invention also provides a method for culturing normal human epidermal melanocyte in a medium of the present invention. According to the method of the present invention, the normal human epidermal melanocyte may be the cells freshly derived from any age and any sexes of human, successively cultured cells of such freshly derived cells, or both of freshly derived and successively cultured cells which are preserved by conventional frozen manner.

As the "cell-culture conditions" of the present invention, any conventional conditions for in vitro cell culturing can be employed and it is well known in the art.

To further illustrate the present invention, and not by way of limitation, the following examples are given.

EXAMPLE OF THE INVENTION Example 1

Normal human epidermal melanocyte culture was performed in modified MCDB 153 with various Ca²⁺ concentrations.

The modified MCDB 153 medium shown in the above table 2 supplemented with the substances shown in following table 3 and fetal bovine serum at a final concentration of 5%(v/v) was used. Final Ca²⁺ concentration of the medium was adjusted with CaCl₂.2H₂ O between 0.15 and 1.2 mM. The final Mg²⁺ concentration of 0.6 mM is same as conventional MCDB 153.

                  TABLE 3     ______________________________________     additives     concentration (final)     ______________________________________     BPE                0.2%    (v/v)     Insulin            10      μg/ml     PMA                20      ng/ml     bFGF               2       ng/ml     Heparin            1       μg/ml     hydrocortisone     0.5     μg/ml     transferrin        1       μg/ml     gentamicin sulfate 50      μg/ml     amphotericin B     50      ng/ml     ______________________________________

Normal human epidermal melanocyte obtained from secondarily cultured cells and stored frozen at liquid nitrogen temperature (Cascade Biologics Inc.) were used. The culture of the cells was performed by using 12-well cell culture plate (Corning). After thawing, the cells were inoculated into the respective media at an initial cell density of 5000 cells/cm². Cells were incubated at 37° C. in 5% CO₂ humidified incubator. Cultures were fed fresh media every or every other day. After 7 days incubation, cells were harvested with 0.025% trypsin/0.01% EDTA, and cell numbers were counted. Cell numbers were determined with hemocytometer counting chamber. Results are shown in FIG. 1. Improved cell growth was shown with the medium containing 0.6-0.9 mM of Ca²⁺.

Example 2

The modified MCDB 153 medium shown in the above table 2 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) was used. Final Ca²⁺ concentration was adjusted to 0.15 mM (medium 1) and 0.9 mM (medium 2).

Normal human epidermal melanocyte obtained from secondarily cultured cells and stored frozen at liquid nitrogen temperature (Cascade Biologics Inc.) were used. The culture of the cells was performed by using 12-well cell culture plate (Corning). After thawing, the cells were inoculated into the respective media at an initial cell density of 5000 cells/cm². Cells were incubated at 37° C. in 5% CO₂ humidified incubator. Cultures were fed fresh media every or every other day.

After 7 days incubation, cells were harvested with 0.025% trypsin/0.01% EDTA, and obtained cell (3rd generation culture) numbers were determined with hemocytometer counting chamber.

Then, the obtained cells were washed and inoculated into freshly prepared respective media at an cell density of 5000 cells/cm². The cells were incubated for additional 7 days and then cell numbers of the culture (4th generation culture) were determined as above. Results are shown in FIG. 2.

Improved cell proliferation were observed with the Ca²⁺ enriched medium both of 3rd and 4th generation of cultures.

Example 3

In to the media of example 2, containing Ca²⁺ at final concentrations of 0.15 mM and 0.9 mM respectively, various amounts of MgCl₂ 6H₂ O were added to provide final Mg²⁺ concentration from 0.6 to 6 mM. Normal human epidermal melanocyte culture was done with said media according to the procedure of example 1. After 7 days incubation, cells were harvested and cell numbers were determined. The results are shown in FIG. 3.

Cell morphology on light microscope in the different culture conditions were shown in FIGS. 4-8. With a modified MCDB 153 containing 0.15 mM of Ca²⁺ and 0.6 mM of Mg²⁺, almost all cells were shown to be bipolar morphology, i.e. having only two ramifications per cell (FIG. 4). With a medium enriched only Ca²⁺ or Mg²⁺, cells were shown to be bipolar morphology, too (FIGS. 5 and 6). In contrast, with media enriched both of Ca²⁺ and Mg²⁺, high population of the cultured cells were shown to be dendrites (FIGS. 7 and 8). In addition, cells cultured with such media were shown comparatively deep color indicating increased intracellular melanin synthesis and deposition (FIGS. 7 and 8).

Example 4

A basal medium shown in following table 4 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) were used. Final Ca²⁺ and Mg²⁺ concentrations were adjusted according to table 5 respectively.

Normal human epidermal melanocyte were cultured using the above media according to the procedure of example 1. After 5 days incubation, the cells were harvested and cell numbers were determined. The results are shown in FIG. 9.

                  TABLE 4     ______________________________________     Components of basal medium of example 4     Component          mg/l     ______________________________________     L-Alanine          4.45     DL-α-Alanine 25     L-Arginine HCl     140.0     L-Asparagine.H.sub.2 O                        7.51     L-Aspartic Acid    2.00     DL-Aspartic Acid   30     L-Cysteine HCl.H.sub.2 O                        21.26     L-Cystine          10     L-Glutamic Acid    7.36     DL-Glutamic Acid.H.sub.2 O                        75     L-Glutamine        488.6     Glycine            28.76     L-Histidine HCl.H.sub.2 O                        43.5     L-Isoleucine       50.15     DL-Isoleucine      20     L-Leucine          32.8     DL-Leucine         60     L-Lysine HCl       44.14     L-Methionine       8.96     DL-Methionine      15     L-Phenylalanie     9.91     DL-Phenylalanine   25     L-Proline          32.27     Hydroxy-L-Proline  5     L-Serine           31.53     DL-Serine          25     L-Threonine        5.96     DL-Threonine       30     L-Tryptophan       6.13     DL-Tryptophan      10.00     L-Tyrosine         28.16     L-Valine           17.57     DL-Valine          25     p-Amino Benzoic acid                        0.025     Ascorbic Acid      0.025     d-Biotin           0.012     Folic Acid         0.4     DL-α-Lipoic Acid                        0.103     Nicotinamide       0.0308     Nicotinic Acid     0.0125     Ethanolamine       3.05     Phosphoethanolamine                        7.05     D-Pantothenate(Ca) 0.239     Pyridoxal.HCl      0.0125     Pyridoxine.HCl     0.0434     α-tocopherol phosphate.2Na                        0.005     Riboflavin         0.376     Thiamin HCl        0.174     Vitamin A          0.05     Vitamin B.sub.12   2.04     Vitamin B.sub.2    0.05     Vitamin K.sub.3    0.005     Adenine sulfate    5     Adenine            12.2     AMP                0.1     ATP-2Na            0.5     Cholesterol        0.1     Deoxyribose        0.25     Choline Chloride   7.23     D-Glucose          1041     myo-lnositol       9.035     Glutathione        0.025     Guanine.HCl        0.15     Hypoxanthine       0.15     Xanthine           0.15     Ribose             0.25     Thymine            0.15     Tween 80           10     Uracil             0.15     Putrescine.2HCl    0.0806     Sodium Pyruvate    27.5     Thymidine          0.363     CaCl.sub.2.2H.sub.2 O                        11.03     CaCl.sub.2         70     KCl                256     MgCl.sub.2.6H.sub.2 O                        61     MgSO.sub.4.7H.sub.2 O                        100     NaCl               7800     Na.sub.2 HPO.sub.4.7H.sub.2 O                        268     Na.sub.2 HPO.sub.4.2H.sub.2 O                        30     KH.sub.2 PO.sub.4  30     CuSO.sub.4.5H.sub.2 O                        0.00138     FeSO.sub.4.7H.sub.2 O                        0.695     Fe(NO.sub.3).sub.3 0.05     H.sub.2 SeO.sub.3  0.00193     MnSO.sub.4.5H.sub.2 O                        0.000121     Na.sub.2 SiO.sub.3.9H.sub.2 O                        0.0711     (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                        0.000618     NH.sub.4 VO.sub.3  0.000293     NiCl.sub.2.6H.sub.2 O                        0.0000595     SnCl.sub.2.2H.sub.2 O                        0.0000565     ZnSO.sub.4.7H.sub.2 O                        0.0719     HEPES              3336     NaHCO.sub.3        1213     Sodium Acetate 3H.sub.2 O                        250     Sodium Acetate     25     Phenol Red(Na)     10     ______________________________________

                  TABLE 5     ______________________________________     Final concentrations of Ca.sup.2+  and Mg.sup.2+     medium No. 1      2         3    4     5   6     ______________________________________     Ca.sup.2+  (mM)                0.95   0.95      0.95 1.1   1.1 1.1     Mg.sub.2+  (mM)                0.4    0.7       5.5  0.4   0.7 5.5     ______________________________________

Example 5

A basal medium shown in following table 6 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) was used. Final Ca²⁺ and Mg²⁺ concentrations were adjusted according to table 7 respectively.

Normal human epidermal melanocyte were cultured using the above media according to the procedure of example 1. After 5 days incubation, the cells were harvested and cell numbers were determined. The results are shown in FIG. 10.

                  TABLE 6     ______________________________________     Components of basal medium of example 5     Component        mg/l     ______________________________________     L-Alanine        8.96     L-Arginine.HCl   210.9     L-Asparagine.H.sub.2 O                      15.01     L-Aspartic Acid  8.50     L-Cysteine HCl.H.sub.2 O                      20.01     L-Cystine        12.5     L-Glutamic Acid  14.71     L-Glutamine      511.6     Glycine          7.51     L-Histidine HCl.H.sub.2 O                      45.0     L-Isoleucine     51.45     L-Leucine        39.3     L-Lysine.HCl     23.64     L-Methionine     11.20     L-Phenylalanie   12.41     L-Proline        23.02     L-Serine         36.78     L-Threonine      7.74     L-Tryptophan     6.43     L-Tyrosine       9.06     L-Valine         19.32     d-Biotin         0.019     Folic Acid       1.06     DL-alpha-Lipoic Acid                      0.203     Nicotinamide     0.326     Ethanolamine     3.05     Phosphoethanolamine                      7.05     D-Pantothenate(Ca)                      0.592     Pyridoxine.HCl   0.134     Riboflavin       0.376     Thiamin HCl      0.669     Vitamin B.sub.12 2.72     Adenine          12.2     Choline Chloride 7.33     D-Glucose        1091     myo-lnositol     9.281     Hypoxanthine     2     Putrescine.2HCl  0.0806     Sodium Pyruvate  82.5     Thymidine        0.713     CaCl.sub.2.2H.sub.2 O                      33.08     KCl              198     MgCl.sub.2.6H.sub.2 O                      61     MgSO.sub.4.7H.sub.2 O                      76.4     NaCl             7500     Na.sub.2 HPO.sub.4.7H.sub.2 O                      268     Na.sub.2 HPO.sub.4                      76.9     KH.sub.2 PO.sub.4                      41.5     CuSO.sub.4.5H.sub.2 O                      0.00263     FeSO.sub.4.7H.sub.2 O                      1.112     H.sub.2 SeO.sub.3                      0.00193     MnSO.sub.4.5H.sub.2 O                      0.000121     Na.sub.2 SiO.sub.3.9H.sub.2 O                      0.0711     (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                      0.000618     NH.sub.4 VO.sub.3                      0.000293     NiCl.sub.2.6H.sub.2 O                      0.0000595     SnCl.sub.2.2H.sub.2 O                      0.0000565     ZnSO.sub.4.7H.sub.2 O                      0.0863     HEPES            3336     NaHCO.sub.3      1188     Sodium Acetate 3H.sub.2 O                      250     Phenol Red(Na)   0.621     ______________________________________

                  TABLE 7     ______________________________________     Final concentrations of Ca.sup.2+  and Mg.sup.2+     medium No.             1       2      3     4    5     6    7     ______________________________________     Ca.sup.2+  (mM)             0.20    0.20   0.20  0.95 0.95  1.15 1.15     Mg.sub.2+  (mM)             0.3     1.25   5.5   1.25 5.5   1.25 5.5     ______________________________________

Example 6

A basal medium shown in following table 8 supplemented with the substances shown in above table 3 and fetal bovine serum at a final concentration of 5%(v/v) were used. Final Ca²⁺ and Mg²⁺ concentrations were adjusted according to table 9 respectively.

Normal human epidermal melanocyte were cultured using the above media according to the procedure of example 1. After 7 days incubation, the cells were harvested and cell numbers were determined. The results are shown in FIG. 11.

                  TABLE 8     ______________________________________     Components of basal medium of example 6     Component        mg/l     ______________________________________     L-Alanine        9     L-Arginine.HCl   211     L-Asparagine.H.sub.2 O                      15.01     L-Aspartic Acid  13     L-Cystine        25     L-Glutamic Acid  14.7     L-Glutamine      146     Glycine          7.51     L-Histidine HCl.H.sub.2 O                      23     L-Isoleucine     2.6     L-Leucine        13     L-Lysine.HCl     29     L-Methionine     4.48     L-Phenylalanie   5     L-Proline        11.5     L-Serine         10.5     L-Threonine      3.57     L-Tryptophan     0.6     L-Tyrosine       1.81     L-Valine         3.5     d-Biotin         0.024     Folic Acid       1.32     DL-α-Lipoic Acid                      0.2     Nicotinamide     0.615     D-Pantothenate(Ca)                      0.715     Pyridoxine.HCl   0.206     Riboflavin       0.0238     Thiamin HCl      1     Vitamin B.sub.12 1.36     Choline Chloride 0.698     D-Glucose        1100     myo-lnositol     0.541     Hypoxanthine     4     Sodium Pyruvate  110     Thymidine        0.7     CaCl.sub.2.2H.sub.2 O                      44.1     KCl              285     MgSO.sub.4.7H.sub.2 O                      152.8     NaCl             7400     Na.sub.2 HPO.sub.4                      153.7     KH.sub.2 PO.sub.4                      83     CuSO.sub.4.5H.sub.2 O                      0.0025     FeSO.sub.4.7H.sub.2 O                      0.834     ZnSO.sub.4.7H.sub.2 O                      0.0288     NaHCO.sub.3      1200     ______________________________________

                  TABLE 9     ______________________________________     Final concentrations of Ca.sup.2+  and Mg.sup.2+     medium No.               1         2      3       4    5     ______________________________________     Ca.sup.2+ 0.3M)     0.95   0.95    1.15 1.15     Mg.sub.2+ 0.65)     0.65   5.5     0.65 5.5     ______________________________________

Example 7

The modified MCDB153 medium shown in above table 2 supplemented with the substances shown in above table 3 was used. The medium contains 0.15 mM of Ca²⁺ and 0.60 mM of Mg²⁺. In addition, the medium containing 0.9 mM of Ca²⁺ and 2.0 mM of Mg²⁺ was prepared. Various amounts of FBS were added into the both of media to yield a final concentration of from 0.001 to 10% (v/v). Normal human epidermal melanocyte were cultured in the above respective media according to the procedure of example 1. After 6 days incubation, the cells were harvested and cell number were determined. The results were shown in FIG. 11.

A maximum proliferation of the cell was obtained With a medium containing 0.01% of FBS. At any concentration of FBS, the cell proliferation obtained with the Ca²⁺ or Mg²⁺ enriched media was superior to that of conventional media. 

What is claimed is:
 1. A medium for culturing normal human epidermal melanocytes in vitro, comprising a basal medium for culturing animal cells, Ca²⁺ at a final concentration of between about 0.9 mM and about 1.2 mM, Mg²⁺ at a final concentration of between about 1.2 mM and about 6.0 mM, and serum at a final concentration of between about 0.001% and about 0.1% (v/v).
 2. The medium of claim 1, further comprising one or more growth factors useful for growth of normal human epidermal melanocytes.
 3. The medium of claim 2, wherein the basal medium for animal cell culture is MCDB
 153. 4. The medium of claim 1, further comprising bovine pituitary extract.
 5. The medium of claim 4, wherein the basal medium for animal cell culture is MCDB
 153. 6. The medium of claim 1, wherein the basal medium for animal cell culture is MCDB
 153. 7. A medium for culturing normal human epidermal melanocyte in vitro, comprising a basal medium for culturing animal cells, Ca²⁺ at a final concentration of between about 0.15 mM and about 1.2 mM, Mg²⁺ at a final concentration of between about 1.2 mM and about 6.0 mM and serum at a concentration of between about 0.001% and about 0.1% (v/v).
 8. The medium of claim 7, wherein the final concentration of Ca²⁺ is between about 0.9 mM and about 1.2 mM and the final concentration of Mg²⁺ is between about 1.2 mM and 6.0 mM.
 9. The medium of claim 7, further comprising one or more growth factors useful for growth of normal human epidermal melanocytes.
 10. The medium of claim 7, further comprising bovine pituitary extract.
 11. The medium of claim 7, wherein the basal medium for animal cell culture is MCDB
 153. 